The following tests were performed on a total of 20 eyes: Schirmer tear test, intraocular pressure (IOP), assessment of conjunctival flora, and pupillary diameter with application of topical tropicamide in 10 healthy captive red kangaroos (Macropus rufus) under manual restraint. The mean Schirmer tear test value was 22.6 ± 6.07 mm/min. The mean intraocular pressure was 17.45 ± 7.23 mm Hg. Values did not differ between eyes or gender for either test, but significant differences were identified for IOP values according to age. The most common bacteria isolated from the conjunctival fornix were Staphylococcus epidermidis (54.5%) and Corynebacterium sp. (18.2%). The mean onset of mydriasis after instillation of 1% tropicamide ophthalmic solution was 16.7 ± 3.34 min and the mean duration of effect was 17.6 ± 8.26 hr. The data obtained in this investigation will aid veterinary ophthalmologists and zoo veterinarians to diagnose ocular diseases in the red kangaroo accurately.